Neutrophils and NADPH Oxidases Are Major Contributors to Mild but Not Severe Ischemic Acute Kidney Injury in Mice.

Upregulation of free radical-generating NADPH oxidases (NOX), xanthine oxidoreductase (XOR), and neutrophil infiltration-induced, NOX2-mediated respiratory burst contribute to renal ischemia-reperfusion injury (IRI), but their roles may depend on the severity of IRI. We investigated the role of NOX, XOR, and neutrophils in developing IRI of various severities. C57BL/6 and Mcl-1ΔMyelo neutrophil-deficient mice were used. Oxidases were silenced by RNA interference (RNAi) or pharmacologically inhibited. Kidney function, morphology, immunohistochemistry and mRNA expression were assessed. After reperfusion, the expression of NOX enzymes and XOR increased until 6 h and from 15 h, respectively, while neutrophil infiltration was prominent from 3 h. NOX4 and XOR silencing or pharmacological XOR inhibition did not protect the kidney from IRI. Attenuation of NOX enzyme-induced oxidative stress by apocynin and neutrophil deficiency improved kidney function and ameliorated morphological damage after mild but not moderate/severe IRI. The IR-induced postischemic renal functional impairment (BUN, Lcn-2), tubular necrosis score, inflammation (TNF-α, F4/80), and decreases in the antioxidant enzyme (GPx3) mRNA expression were attenuated by both apocynin and neutrophil deficiency. Inhibition of NOX enzyme-induced oxidative stress or the lack of infiltration by NOX2-expressing neutrophils can attenuate reperfusion injury after mild but not moderate/severe renal IR.

Cold Saline Perfusion before Ischemia-Reperfusion Is Harmful to the Kidney and Is Associated with the Loss of Ezrin, a Cytoskeletal Protein, in Rats.

BACKGROUND: Organ protection for transplantation is perfusion with ice-cold preservation solutions, although saline is also used in animal experiments and living donor transplantations. However, ice-cold perfusion can contribute to initial graft injury. Our aim was to test if cytoskeletal damage of parenchymal cells is caused by saline itself or by the ice-cold solution. METHODS: F344 rat kidneys were flushed with cold (4 °C) saline, ischemic and sham kidneys were not perfused. In a separate set, F344 kidneys were flushed with saline or preservation solution at 4 or 15 °C. Ischemia time was 30 min. RESULTS: Renal injury was significantly more severe following cold ischemia (CI) than after ischemia-reperfusion without flushing (ischemia/reperfusion (I/R)). Functional and morphologic damage was accompanied by severe loss of ezrin from glomerular and tubular epithelial cells after CI. Moreover, saline caused serious injury independently from its temperature, while the perfusion solution was more beneficial, especially at 4 °C. CONCLUSIONS: Flushing the kidney with ice-cold saline can cause more severe injury than ischemia-reperfusion at body temperature even during a short (30 min) ischemia. Saline perfusion can prolong recovery from ischemia in kidney transplantation, which can be prevented by using preservation solutions.

Glomerular Collagen Deposition and Lipocalin-2 Expression Are Early Signs of Renal Injury in Prediabetic Obese Rats.

Feeding rats with high-fat diet (HFD) with a single streptozotocin (STZ) injection induced obesity, slightly elevated fasting blood glucose and impaired glucose and insulin tolerance, and caused cardiac hypertrophy and mild diastolic dysfunction as published before by Koncsos et al. in 2016. Here we aimed to explore the renal consequences in the same groups of rats. Male Long-Evans rats were fed normal chow (CON; n = 9) or HFD containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 blood and urine samples were taken and kidney and liver samples were collected for histology, immunohistochemistry and for analysis of gene expression. HFD and STZ increased body weight and visceral adiposity and plasma leptin concentration. Despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1ß mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression, and cortical and medullary mRNA expression of TGF-ß1, Nrf2, and PPARγ were similar in CON and PRED rats. Reduced AMPKα phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while plasma lipid and liver enzyme concentrations were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury.

Therapeutic miR-21 Silencing Ameliorates Diabetic Kidney Disease in Mice.

Diabetic nephropathy is the main cause of end-stage renal disease. MicroRNAs are powerful regulators of the genome, and global expression profiling revealed miR-21 to be among the most highly regulated microRNAs in kidneys of mice with diabetic nephropathy. In kidney biopsies of diabetic patients, miR-21 correlated with tubulointerstitial injury. In situ PCR analysis showed a specific enrichment of miR-21 in glomerular cells. We identified cell division cycle 25a (Cdc25a) and cyclin-dependent kinase 6 (Cdk6) as novel miR-21 targets in mesangial cells. miR-21-mediated repression of Cdc25a and Cdk6 resulted in impaired cell cycle progression and subsequent mesangial cell hypertrophy. miR-21 increased podocyte motility by regulating phosphatase and tensin homolog (Pten). miR-21 antagonism in vitro and in vivo in streptozotocin-induced diabetic mice decreased mesangial expansion, interstitial fibrosis, macrophage infiltration, podocyte loss, albuminuria, and fibrotic- and inflammatory gene expression. In conclusion, miR-21 antagonism rescued various functional and structural parameters in mice with diabetic nephropathy and, thus, might be a viable option in the treatment of patients with diabetic kidney disease.

Urine/Plasma Neutrophil Gelatinase Associated Lipocalin Ratio Is a Sensitive and Specific Marker of Subclinical Acute Kidney Injury in Mice.

BACKGROUND: Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia. METHODS: Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression. RESULTS: A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups. CONCLUSIONS: These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.

Oxidative/Nitrative Stress and Inflammation Drive Progression of Doxorubicin-Induced Renal Fibrosis in Rats as Revealed by Comparing a Normal and a Fibrosis-Resistant Rat Strain.

Chronic renal fibrosis is the final common pathway of end stage renal disease caused by glomerular or tubular pathologies. Genetic background has a strong influence on the progression of chronic renal fibrosis. We recently found that Rowett black hooded rats were resistant to renal fibrosis. We aimed to investigate the role of sustained inflammation and oxidative/nitrative stress in renal fibrosis progression using this new model. Our previous data suggested the involvement of podocytes, thus we investigated renal fibrosis initiated by doxorubicin-induced (5 mg/kg) podocyte damage. Doxorubicin induced progressive glomerular sclerosis followed by increasing proteinuria and reduced bodyweight gain in fibrosis-sensitive, Charles Dawley rats during an 8-week long observation period. In comparison, the fibrosis-resistant, Rowett black hooded rats had longer survival, milder proteinuria and reduced tubular damage as assessed by neutrophil gelatinase-associated lipocalin (NGAL) excretion, reduced loss of the slit diaphragm protein, nephrin, less glomerulosclerosis, tubulointerstitial fibrosis and matrix deposition assessed by periodic acid-Schiff, Picro-Sirius-red staining and fibronectin immunostaining. Less fibrosis was associated with reduced profibrotic transforming growth factor-beta, (TGF-ß1) connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL-1a1) mRNA levels. Milder inflammation demonstrated by histology was confirmed by less monocyte chemotactic protein 1 (MCP-1) mRNA. As a consequence of less inflammation, less oxidative and nitrative stress was obvious by less neutrophil cytosolic factor 1 (p47phox) and NADPH oxidase-2 (p91phox) mRNA. Reduced oxidative enzyme expression was accompanied by less lipid peroxidation as demonstrated by 4-hydroxynonenal (HNE) and less protein nitrosylation demonstrated by nitrotyrosine (NT) immunohistochemistry and quantified by Western blot. Our results demonstrate that mediators of fibrosis, inflammation and oxidative/nitrative stress were suppressed in doxorubicin nephropathy in fibrosis-resistant Rowett black hooded rats underlying the importance of these pathomechanisms in the progression of renal fibrosis initiated by glomerular podocyte damage.

LPS-induced delayed preconditioning is mediated by Hsp90 and involves the heat shock response in mouse kidney.

INTRODUCTION: We and others demonstrated previously that preconditioning with endotoxin (LPS) protected from a subsequent lethal LPS challenge or from renal ischemia-reperfusion injury (IRI). LPS is effective in evoking the heat shock response, an ancient and essential cellular defense mechanism, which plays a role in resistance to, and recovery from diseases. Here, by using the pharmacological Hsp90 inhibitor novobiocin (NB), we investigated the role of Hsp90 and the heat shock response in LPS-induced delayed renal preconditioning. METHODS: Male C57BL/6 mice were treated with preconditioning (P: 2 mg/kg, i.p.) and subsequent lethal (L: 10 mg/kg, i.p.) doses of LPS alone or in combination with NB (100 mg/kg, i.p.). Controls received saline (C) or NB. RESULTS: Preconditioning LPS conferred protection from a subsequent lethal LPS treatment. Importantly, the protective effect of LPS preconditioning was completely abolished by a concomitant treatment with NB. LPS induced a marked heat shock protein increase as demonstrated by Western blots of Hsp70 and Hsp90. NB alone also stimulated Hsp70 and Hsp90 mRNA but not protein expression. However, Hsp70 and Hsp90 protein induction in LPS-treated mice was abolished by a concomitant NB treatment, demonstrating a NB-induced impairment of the heat shock response to LPS preconditioning. CONCLUSION: LPS-induced heat shock protein induction and tolerance to a subsequent lethal LPS treatment was prevented by the Hsp90 inhibitor, novobiocin. Our findings demonstrate a critical role of Hsp90 in LPS signaling, and a potential involvement of the heat shock response in LPS-induced preconditioning.

Activation of the miR-17 family and miR-21 during murine kidney ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) is the main cause of acute kidney injury (AKI) in patients. We investigated renal microRNA (miRNA) expression profiles and the time course of changes in selected miRNA expressions after renal I/R to characterize the miRNA network activated during development and recovery from AKI. METHODS AND RESULTS: One day after lethal (30 minutes) and sublethal (20 minutes) renal ischemia, AKI was verified by renal histology (tubular necrosis, regeneration), blood urea nitrogen (BUN) level, renal mRNA expression, and plasma concentration of neutrophil gelatinase-associated lipocalin (NGAL) in C57BL/6J mice. On the first day after 30-minute, lethal I/R miR-21, miR-17-5p, and miR-106a were elevated out of the 21 miRNAs successfully profiled on the Luminex multiplex assay. After 20-minute, sublethal I/R, renal miR-17-5p and miR-106a expressions were elevated on the first and second days of reperfusion, while miR-21 expression increased later and lasted longer. Renal miR-17-5p and miR-21 expressions correlated with each other. Renal function returned to normal on the fourth day after sublethal I/R. CONCLUSIONS: Our results demonstrate that besides miR-21, miR-17-5p, and miR-106a are additionally activated during the maintenance and recovery phases of renal I/R injury. Furthermore, a correlation between renal miR-17-5p and miR-21 expressions warrants further investigation of how they may influence each other and the outcome of renal ischemia-reperfusion injury.

Immune activation and target organ damage are consequences of hydrodynamic treatment but not delivery of naked siRNAs in mice.

Short-interfering RNAs (siRNAs), key mediators of RNA interference comprise a promising therapeutic tool, although side effects such as interferon (IFN) response are still not perfectly understood. Further, delivery to target organs is a major challenge, possibly associated with side effects including immune activation or organ damage. We investigated whether immune activation as a consequence of double-stranded RNA induced IFN response (Jak/STAT pathway activation or cytokine production) or target organ damage is induced by in vivo low-volume (LV) or high-volume (HV) hydrodynamic delivery or treatment with naked siRNA. NMRI mice were injected with naked siRNAs or saline by hydrodynamic injection (HDI) and positive control mice received polyinosinic-polycytidilic acid (poly I:C). LV (1 mL/mouse) and HV (10% of body weight) HDI were compared. After LV HDI, STAT1 and OAS1 gene expression inflammatory cytokine plasma levels and target organ injury were assessed. LV HDI induced slight alanine aminotransferase elevation and mild hepatocyte injury, whereas HV HDI resulted in high ALAT level and extensive hepatocyte necrosis. STAT1 or OAS1 was not induced by LV siRNA; however, HV saline led to a time-dependent slight increase in gene expression. Inflammatory cytokine plasma level and organ histology and functional parameters demonstrated no damage following LV HDI with or without siRNA. Our data demonstrate that naked siRNAs may be harnessed, without the induction of IFN response or immune activation, and that LV HDI is preferable, because HV HDI may cause organ damage.

Proteomic identification of vanin-1 as a marker of kidney damage in a rat model of type 1 diabetic nephropathy.

At present, the urinary albumin excretion rate is the best noninvasive predictor for diabetic nephropathy (DN) but major limitations are associated with this marker. Here, we used in vivo perfusion technology to establish disease progression markers in an animal model of DN. Rats were perfused with a reactive ester derivative of biotin at various times after streptozotocin treatment. Following homogenization of kidney tissue and affinity purification of biotinylated proteins, a label-free mass spectrometry-based proteomic analysis of tryptic digests identified and relatively quantified 396 proteins. Of these proteins, 24 and 11 were found to be more than 10-fold up- or downregulated, respectively, compared with the same procedure in vehicle-treated rats. Changes in the expression of selected differentially regulated proteins were validated by immunofluorescence detection in kidney tissue from control and diabetic rats. Immunoblot analysis of pooled human urine found that concentrations of vanin-1, an ectoenzyme pantetheinase, distinguished diabetic patients with macroalbuminuria from those with normal albuminuria. Uromodulin was elevated in the urine pools of the diabetic patients, regardless of the degree of albuminuria, compared with healthy controls. Thus, in vivo biotinylation facilitates the detection of disease-specific changes in the abundance of potential biomarker proteins for disease monitoring and/or pharmacodelivery applications.

Elevated systemic TGF-beta impairs aortic vasomotor function through activation of NADPH oxidase-driven superoxide production and leads to hypertension, myocardial remodeling, and increased plaque formation in apoE(-/-) mice.

The role of circulating, systemic TGF-beta levels in endothelial function is not clear. TGF-beta(1) may cause endothelial dysfunction in apolipoprotein E-deficient (apoE(-/-)) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-mo-old control (C57Bl/6), apoE(-/-), TGF-beta(1)-overexpressing (TGFbeta(1)), and crossbred apoE(-/-) x TGFbeta(1) mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-mo-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-beta(1) levels (ELISA) were elevated in TGFbeta(1) mice without downregulation of TGF-beta-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in apoE(-/-) x TGFbeta(1) mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the apoE(-/-) x TGFbeta vs. C57Bl/6 mice. Our results demonstrate that elevated circulating TGF-beta(1) causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in apoE(-/-) mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGFbeta(1).

The Rowett rat strain is resistant to renal fibrosis.

BACKGROUND: Genetic susceptibility to renal fibrosis may determine the individual rate of progression to renal failure. We aimed to study the progression in Rowett (RO) rats, a strain we found resistant to subtotal nephrectomy (SNX), comparing to Sprague-Dawley (SD) rats, a strain with established sensitivity in a radical ablation/infarction and diet-induced SNX model. METHODS: Eight-week-old male RO (RO-SNX) and SD (SD-SNX, n = 5/group) rats underwent SNX and were kept on high protein and salt diet. Kidney function was monitored and the kidneys were evaluated by histology and immunohistochemistry 5 weeks after SNX. RESULTS: RO-SNX rats had only mild proteinuria and less glomerulosclerosis, accompanied by less fibronectin and TGF-beta staining as compared to SD-SNX rats. Glomerular nitrotyrosine staining was less intense in RO-SNX vs SD-SNX, accompanied by less podocyte damage as demonstrated by desmin staining. CONCLUSION: Our results demonstrate the importance of podocyte damage in glomerulosclerosis and that Rowett rats are protected from renal fibrosis. To our knowledge, this is the first strain of rats with unknown genetic resistance, which makes the strain attractive for studying the genetic background of renal fibrosis.

Increased renoprotection with ACE inhibitor plus aldosterone antagonist as compared to monotherapies--the effect on podocytes.

BACKGROUND: Blockade of the renin-angiotensin-aldosterone system (RAAS) does not completely prevent progression of renal disease. Mineralocorticoid receptor blockade provides additional renoprotection over ACE-inhibition monotherapy. We examined the mechanisms underlying superior renoprotection in the subtotal nephrectomy (SNX) model. METHODS: Sprague-Dawley rats were randomized into six groups: (1) sham-op, (2) SNX without treatment, (3) SNX + quinapril (Q), (4) SNX + spironolactone (S), (5) SNX + combination therapy (Q+S), (6) SNX + combination hydrochlorothiazide + reserpin + hydralazine (HRH). Albuminuria and blood pressure were monitored, and kidneys were examined by morphometric and molecular methods. RESULTS: In SNX rats, albumin excretion was significantly higher than in sham-op rats. Blood pressure reduction was not significantly different between the treatment groups. All therapies (S, Q, Q+S and HRH) reduced albuminuria; the values were lowest in animals treated with Q+S. The volume density of glomerular matrix and the number of mesangial cells were significantly increased in SNX and were lowest in SNX treated with Q+S. The number of podocytes was reduced in SNX, but was normalized in SNX treated with Q+S. Glomerular volumes and podocyte volumes were significantly higher in SNX than in sham-op. Both volumes were reduced by all interventions, but almost normalized by treatment with Q+S. Expression of collagen IV, TGF-beta(1) and desmin was increased after SNX and significantly reduced by treatment with Q and Q+S. CONCLUSIONS: In subtotally nephrectomized rats, mineralocorticoid blockade provided additional renoprotection over and above ACE inhibition. Such benefit was paralleled by major changes in podocyte number and morphology and was not blood pressure dependent.

Role of invariant natural killer T (iNKT) cells in systemic lupus erythematosus.

Natural killer T (NKT) cells represent a unique T cell lineage. The NKT cells bearing an invariant TCR (iNKT cells) recognize a small variety of glycolipid antigens in the context of CD1d (non-classical MHC-I) presentation. CD1d-restricted iNKT cells play a regulatory role during an immune response by producing cytokines (IFN-gamma, and IL-4). The identification of alpha-galactosyl-ceramide (alpha-GalCer), a marine sponge derivative as a potent stimulator of iNKT cells has raised the potential of therapeutic iNKT cell activation. Invariant NKT cells have been implicated in several different autoimmune diseases in mice and humans, including systemic lupus erythematosus (SLE). Abnormalities in the number and functions of NKT cells have been observed in SLE patients and mouse strains genetically predisposed to lupus (MRL/lpr, NZB/W F1). Moreover, inverse correlation between the frequency of NKT cells and IgG levels has been observed. Elevated IgG levels in relatives of patients with lupus as well as in patients with lupus were associated with low frequencies of NKT cells. This review focuses on the potential roles of NKT cells in the pathogenesis of SLE. It summarizes recent advances in glycolipid therapy for murine lupus. First, it has been demonstrated, that repeated administration of alpha-GalCer to MRL/lpr mice alleviated inflammatory dermatitis but did not influence kidney disease. Treatment of NZB/W mice with alpha-GalCer resulted in amelioration of SLE symptoms in young mice, but treatment of older animals resulted in disease exacerbation. The effects of NKT cell activation using alpha -GalCer, on disease progression, were influenced by a variety of parameters, including the genetic background of mice, the alpha -GalCer dose, number of injections and the stage of the disease process when treatment was performed. Manipulation of NKT cells in the human system may be a promising treatment alternative for the future, however possible deleterious effects have to be carefully investigated first.

Mechanisms behind flare of renal lupus during murine pregnancy.

The outcome of pregnancy in systemic lupus erythematosus is still controversial. The authors recently reported the disappearance of the manifestation of the skin disease but a diminished survival rate in lupus-prone animals undergoing several pregnancies. It was postulated that lupus-prone animals must have subclinical renal symptoms at an early age and that immune and hormonal changes during pregnancy exacerbate immune reactions in the kidneys, leading to a shortened life span. Here, the authors analysed changes at day 14 of pregnancy in lupus-prone LPR (MRL/lpr) mice and MRL controls regarding cytokines, regulatory T (Treg) cells and deposition of immunocomplexes. Worsened kidney function was observed during pregnancy, even in the absence of lupus signs. This was accompanied by renal inflammation and higher interferon-gamma and interleukin-10 levels. C3 and immunoglobulin G deposition was enhanced in kidney and placenta from lupus-prone pregnant animals. Pregnancy enhanced the levels of Treg cells in control animals but not in lupus-prone animals. As pregnancy-induced Treg cells were shown to be specific for paternal antigens it is not to be expected that these Treg cells can help to destroy autoreactive cells. The authors conclude that early subclinical kidney disease in lupus-prone animals exacerbates during pregnancy. Albeit obtained with an experimental animal model, their data are potentially of importance for lupus patients of reproductive age.